Fig. 6
CircZFR promotes CDK2/cyclin-E1 and CDK2/SSBP1 assembly and interacts with SSBP1. a. Model of in vivo circRNA pull-down using MS2-tagging system, and the following label-free mass spectrometric (MS) analysis. b. Confirmation of the overexpression of circZFR using qRT-PCR analysis. c. Co-transfection of circZFR-MS2GFP and MS2-CP-FlagmCherry plasmids to induce the expression of MS2 RNA hairpins with overexpressed circZFR and a fusion protein MS2-CP-Flag, which could recognize MS2 RNA hairpins (Scale bar = 200 μm). The green fluorescence-labeled circZFR (up) and the red fluorescence-labeled MS2-CP-Flag (bottom). d. Western Blot test the MS2-CP-Flag pulled down by anti-Flag (up). The enrichment of circZFR in the complex with MS2-CP-Flag was detected by qRT-PCR (bottom). The assays were done twice in triplicate. e. The protein complex with MS2-CP-Flag was tested using a label-free MS. The peptides were matched to SSBP1. f. circRNA immunoprecipitation (circRIP) assay to measure the amount of circZFR pulled down by SSBP1 and IgG antibodies in the HeLa and SiHa cells stably overexpressed circZFR and circ-Ctrl. The assays were done twice in triplicate. g. The lysates prepared from the HeLa cells stably overexpressing circZFR, or circZFR knockdown (sh-circ) and the corresponding controls (circ-Ctrl, and sh-Ctrl, respectively) were subjected to immunoprecipitation using anti-CDK2. **P< 0.001, ****P< 0.0001, MS2-CP: MS2 bacteriophage coat protein, MS: mass spectrometric, GFP: green fluorescent protein, circRIP: circRNA immunoprecipitation